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Author Ho, Wen-Zhe
Source CiteSeerX
Content type Text
File Format PDF
Subject Domain (in DDC) Computer science, information & general works ♦ Data processing & computer science
Subject Keyword Human Immune Cell ♦ Real-time Reverse Transcriptase Pcr Assay ♦ Sp Mrna ♦ Sp Real-time Rt-pcr Assay ♦ Substance Mrna ♦ Fetal Brain ♦ Intra-assay Variation ♦ Sp Gene Transcript ♦ Reaction Mixture ♦ Threshold Cycle Number ♦ Reproducible Data ♦ Sp Mrna Level ♦ Peripheral Blood Lymphocyte ♦ Mrna Copy ♦ Real-time Rt-pcr Assay ♦ Major Modulator ♦ Dynamic Range ♦ Sp-specific Primer Pair ♦ Real-time Rt-pcr ♦ Human Blood Monocyte-derived Macrophage ♦ Real-time Reverse Transcriptase ♦ Sp Real-time Pcr Assay ♦ Molecular Beacon ♦ Clinical Investigation ♦ Sp Precursor ♦ Sp Gene ♦ Gene Transcript ♦ Nonneuronal Cell ♦ Specific Probe ♦ Widespread Application ♦ Mrna Expression
Abstract We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quanti-fication of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.
Educational Role Student ♦ Teacher
Age Range above 22 year
Educational Use Research
Education Level UG and PG ♦ Career/Technical Study
Publisher Date 2001-01-01